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Abstract:

The present invention relates to methods for determining whether blood
flow is restricted in a blood vessel of an individual suspected of
compromised blood flow in the vessel, the method comprising the steps of
delivering UTP, a derivative thereof, or a salt thereof to the vessel,
assessing blood flow quantitatively in the vessel by obtaining a value
that correlates to blood flow in said vessel, comparing the obtained
value with a reference value, and determining whether the individual has
compromised blood flow based on the results of the comparison. The
invention also provides for methods of diagnosing atherosclerotic and
ischemic heart diseases using UTP, a derivative thereof, or a salt
thereof, as well as methods for inducing maximal hyperemia for diagnostic
purposes.

31. The method of claim 28, wherein the UTP, the derivative thereof, or
the pharmaceutically acceptable salt of UTP or the derivative is
administered by a FFR thermodilution catheter, a microinfusion catheter
or a guiding catheter.

36. The method of claim 18, wherein the UTP, derivative thereof, or
pharmaceutically acceptable salt of UTP or the UTP derivative is
delivered via catheter.

37. The method of claim 36, wherein the catheter is a FFR/thermodilution
catheter, a microinfusion catheter, a guide catheter or an intravenous
catheter or a combination thereof.

38. The method of claim 18, further comprising measuring one or more of
FFR, CFR, MAP and APV and relative blood flow.

39. The method of claim 18, wherein the UTP, derivative thereof, or
pharmaceutically acceptable salt of UTP or the UTP derivative is
delivered to a blood vessel, the method further comprising assessing
blood flow quantitatively in the vessel by obtaining a value that
correlates to blood flow in said vessel.

40. The method of claim 39, further comprising determining whether the
individual has compromised blood flow by comparing the obtained value
with a reference value.

Description:

CROSS-REFERENCE TO PRIOR APPLICATION

[0001] This application claims the benefit of U.S. Provisional application
Ser. No. 61/232,518, filed Aug. 10, 2009 and Ser. No. 61/357, 857 filed
Jun. 23, 2010, both of which are incorporated by reference herein in
their entirety.

FIELD OF INVENTION

[0002] The present invention relates to methods for determining whether
blood flow is restricted in a blood vessel of an individual suspected of
compromised blood flow in the vessel using UTP, a derivative thereof, or
a salt thereof. Also, it relates to compositions containing UTP, a
derivative thereof, or a salt thereof for use as a diagnostic agent in
the foregoing methods.

BACKGROUND OF INVENTION

[0003] The potent and widespread vascular actions of purine nucleotides
and nucleosides have long been recognized. Naturally occurring
extracellular purine nucleotides and nucleosides exert cardiovascular
responses by stimulating various P1 and P2 receptors [1,2]. Adenine
nucleotides and nucleosides are used for many diagnostic and treatment
purposes in daily clinical practice such as assessment of coronary blood
flow [3-8] and as anti-arrhythmic agents. Adenosine non-selectively
activates 4 receptor subtypes: A1, A2A, A2B, and A3. Activation of
cardiac A2A and A2B adenosine receptors vasodilates the coronary and
peripheral arterial beds, increases myocardial blood flow (MBF), and
causes sympathoexcitation, but also results in mast cell degranulation
and bronchial constriction. Nevertheless, intracoronary and intravenous
adenosine are employed in the clinic for assessment of fractional flow
reserve (FFR).

[0004] Uridine 5-triphosphate (UTP) is also a naturally occurring compound
in the circulation and is discharged during acute myocardial infarction.
UTP stimulates P2Y2 and P2Y4 receptors, where the first are predominant
in the human cardiovascular tree. UTP is highly selective for this
receptor.

[0005] Previous assumptions underlying the use of UTP in treatment of
cardiovascular disease have proved inaccurate in vivo. It was
demonstrated in 2004 and 2008 [9,10], by using local infusion of ATP and
UTP, that these two agents were the only registered metabolites capable
of opposing sympathetic vasoconstriction and concomitantly increasing
blood flow (85% and 60% of maximum in the leg during hard exercise,
respectively). However, the present inventor, using systemic infusion of
UTP in pigs, has found that UTP can only maximally lower mean arterial
pressure by 30% (Example 4), which is substantially less than that
achieved by ATP because ATP can produce limitless lowering of blood
pressure. Therefore, ATP is more potent than UTP systemically as well as
locally in the healthy leg, although both ATP and UTP were shown
previously to be equipotent in the peripheral vascular system for the
P2Y2 receptor in the arms of humans [11].

[0006] The higher potency of ATP compared to UTP in the normal healthy leg
was attributed to the degradation of ATP to ADP, AMP, and adenosine.
These degradation products, in conjunction with ATP, could contribute to
elevate blood flow higher than that achieved by UTP, which does not have
any vasoactive degradation products. Thus, a comparison of the relative
vasoactive potencies of exogenous nucleotides and adenosine revealed the
following rank order: ATP (100)=UTP (100)>>adenosine (5.8)>ADP
(2.7)>AMP (1.7), but only for blood flows around 3.5 Lmin-1 [9].

[0007] Comparative studies in the human leg of healthy and diabetic
patients showed that UTP>>ATP with regard to vasodilation in legs
of elderly patients and patients with type 2 diabetes [12]. This is
clearly in contrast to previous findings, but is more clinically relevant
as most cardiac patients are older. Interestingly, the discrepancy in
vasoactive potency was not due to an up-regulation of the P2Y2 receptor,
suggesting that up-regulation of other ATP-related (but not UTP)
vasoconstrictive receptors must be relevant when people are of advanced
age and poor general health. However, recent studies have shown that the
ability to oppose sympathetic vasoconstriction during exercise is intact
in patients with type 2 diabetes, suggesting that previous assumptions of
a crucial involvement of the P2Y2 receptor in functional sympatholysis
are inaccurate [9]. This conclusion is also supported by another study by
the present inventor in pigs where ADP or UTP was infused during
myocardial infarction. In that study, UTP increased the infarct area
during acute myocardial infarction, whereas ADP diminished it
(unpublished results). This suggests a pharmacological cardioprotective
effect of ADP, but a detrimental effect of UTP, suggesting that caution
must be exercised when using UTP in clinically acute conditions.
Therefore, the previous assumptions in patent application WO 2007/065437
relating to the regulation of purinergic receptor activity to modulate
vascular tone, particularly for treating hemodynamic conditions by
overriding vasoconstriction activity, which could potentially have
clinical applications for treatment with UTP agonists or antagonists, is
incorrect.

[0008] In contrast to the vasodilating properties associated with UTP
mentioned above, UTP has also been described to be a potent
vasoconstrictor in the coronary circulation. Of particular note are the
studies relating to human coronary arteries and bypass vessels [13-15].
In these studies, both UTP and UTP.sub.γS induced contractions in
coronary arteries and the internal mammary artery in heart transplant
patients, suggesting that P2Y2 receptors are important contractile
receptors. UTP.sub.γS also induced contractions in the saphenous
vein. Given that prolonged treatment with UTP has been shown to induce
smooth muscle proliferation in vitro, it can be speculated that in case
of endothelial dysfunction (such as with coronary artery disease),
extracellular nucleotides derived from the blood may reach smooth muscle
cells (SMCs), leading to UTP-mediated vasoconstriction of P2Y2 receptors.
In human coronary arteries, the P2Y2-subtype has been presumed to play a
major role in this speculated constriction [14,15]. This is also seen in
animal studies [16,17]. In pigs, the P2Y2 receptor is up-regulated in
SMCs of in vivo stented coronary arteries to mediate the mitogenic
effects of nucleotides [18]. Therefore, the P2Y2 receptors are suspected
to take part in the pathophysiological genesis of potentially
life-threatening vasospasms [15].

[0009] Besides being looked at as evoking coronary vasoconstriction of
damaged vessels, extracellular nucleotides have also been implicated to
play an important role in the development of inflammatory vascular
disease [19,20]. Seye et al. showed that UTP, acting at P2Y2 receptors,
promoted intimal hyperplasia of collared rabbit carotid arteries [21]. In
an animal model, porcine P2Y2 receptors were found to be overexpressed in
stented coronaries and to play a distinct mitogenic role there [18]. It
has thus been accepted that vascular remodeling, facilitated by
extracellular nucleotides, is a key step in the genesis of cardiovascular
and cerebrovascular disease, potentially culminating in life-threatening
states of stroke or heart attack. Therefore, no preceding clinical
studies have ever been attempted involving in vivo coronary infusion of
UTP in humans, because this compound, for all the above mentioned
reasons, was believed to be hazardous to humans.

[0010] Angiographic assessment of coronary artery disease (CAD) has guided
cardiac therapy for more than 30 years, however even experienced
angiographers are unable to reliably assess lesion severity because
angiography has significant intra-observer and inter-observer variability
and is not a physiological assessment, but merely a visual one. Recent
studies, such as the COURAGE trial, have re-emphasized what all current
medical guidelines recommend: that for low risk patients, even those
experiencing angina, optimal medical therapy should be the initial
treatment. For those patients whose disease progresses, or for whom chest
pain is not alleviated, revascularization, either through angioplasty and
stenting or surgery, should be performed.

[0011] The new diagnostic tool fractional Flow Reserve (FFR) helps
physicians to decide whether to intervene on a stenosis (i.e., abnormal
narrowing of blood vessels) or not. Achievement of maximal hyperemia of
coronary microcirculation is a prerequisite for the exact assessment of
FFR in order to minimize the effect of microvascular resistance. Thus,
the higher the flow, the larger the pressure drop across the stenotic
vessel, i.e., the lower the FFR. For accurate FFR measurements,
achievement of maximal hyperemia is imperative for minimizing the effect
of microvascular resistance. Only at maximal hyperemia is flow and
pressure linearly correlated. If there is only suboptimal hyperemia, the
FFR index underestimates the functional severity of coronary stenosis.
This can lead to injurious outcomes [22]. Therefore, it is crucial for
clinical decision making, that the FFR response is accurate, otherwise
over- or under-treatment of patients will occur, leading to higher
mortality rates and more expensive treatment regimens.

[0012] The preferred standard hyperemic agent used today for inducing
coronary hyperemia is adenosine. However, adenosine use is associated
with side effects even with local infusions. For example, adenosine
causes dyspnea and angina in nearly all patients, as well as
second-degree AV block in some patients. Adenosine use is also associated
with contraindications such as asthma, COPD, angina, hypotension,
2nd or 3rd-degree AV block, and sinus node dysfunction; and the
need for abstinence from caffeine in order to get an accurate hyperemic
assessment, because caffeine blocks P1 receptors, which are the
vasodilatatory receptors the adenine compounds function through [23].

[0013] Given the foregoing limitations associated with the use of
adenine-related compounds (e.g., adenosine and ATP) as hyperemic agents,
more potent hyperemic agents with fewer side effects would be beneficial
for diagnosing compromised blood flow in blood vessels.

SUMMARY OF THE INVENTION

[0014] The present invention is predicated on the finding of the present
inventor that UTP is a better vasodilator than other clinically used
vasodilators: adenosine, ATP and NO, which are all equipotent in
situations where patients suffer from coronary artery disease. Thus,
although UTP may not be used as a therapeutic because it rapidly
desensitizes P2Y2 receptors, may cause endothelial cell proliferation
which leads to atherosclerosis during prolonged exposure, and has too
short a terminal half-life (20 seconds), it can surprisingly be used as
an optimal diagnostic agent. This is supported by the evidence (presented
in Examples 2 and 3) in humans, who were being evaluated for potential
coronary atherosclerosis, showing the much higher potency of UTP:
UTP>>ado (=ATP) in terms of vasodilator activity. In this study, it
was shown that UTP more effectively lowers FFR than adenosine, allowing
for more accurate maximal coronary hyperemia to be achieved. This was the
first use of UTP in the context of in vivo coronary circulation. In fact,
previous in vitro studies demonstrated that UTP induces vasoconstriction,
which would diminish coronary perfusion. Thus, the finding that UTP can
induce hyperemia in patients with suspected endothelial dysfunction is
surprising.

[0015] In contrast to ATP, UTP is highly receptor selective and degraded
rapidly (terminal half-life: about 20 sec) in the circulation, rendering
an inactive degradation product. Thus, it has no long-term effects. UTP
can easily be applied to patients that are in a stable clinical condition
or during recurrent angina attacks. Surprisingly, the present inventor
also found that UTP is a more potent vasodilator than adenosine and ATP
in both peripheral circulation of diabetics and in the coronary
circulation of patients with coronary artery disease. Thus, UTP induces
maximal hyperemia to a greater extent than both adenosine and ATP. This
is an important finding given that maximal hyperemia is important, for
example, in the exact measurement of FFR in order to minimize the effect
of microvascular resistance.

[0016] The advantages of using UTP as a diagnostic therefore include its
specific affinity for the P2Y2 receptor, which makes it more receptor
selective than other diagnostic agents, UTP (unlike ATP) does not have a
degradation product which is vasoactive, and no abstinence from caffeine
is required for accurate hyperemic assessment. It reaches the steady
state faster and acts rapidly (time to peak, 5 s), it is easy to use,
lacks significant side effects, has a terminal half-life of 20 s, has no
obvious contraindications, and can be used for patients who have
contraindications to the use of adenosine due to, for example,
arrhythmia, COPD, or asthma. For the foregoing reasons, UTP is an ideal
hyperemic agent for use in diagnosing compromised blood flow in blood
vessels.

[0017] The inventor has found that UTP, a derivative thereof, or a salt
thereof (as further defined below), can be used to determine whether
blood flow is restricted in a blood vessel of an individual suspected of
compromised blood flow in the vessel, by mimicking the increased blood
flow observed during exercise. Accordingly, compromised blood flow can be
determined with high accuracy even in individuals at rest. Thus, the
method is useful for determining compromised blood flow in any
individual, even in individuals who should not or for whom it is
undesirable to undergo exercise testing.

[0018] The present invention is believed to represent the first diagnostic
use of UTP, a derivative thereof, or a salt thereof. Consequently, in one
aspect, the invention relates to compositions containing an effective
amount of UTP, a derivative thereof or a salt thereof for use as a
diagnostic agent in assessing blood flow.

[0019] By determining that blood flow is compromised in a blood vessel,
preferably an artery of an individual with suspected compromised blood
flow, hemodynamic conditions caused by compromised blood flow,
particularly but not exclusively stenosis, may be treated more
effectively. In particular, stenosis may be recognised before any adverse
cardiovascular events, such as myocardial infarction, stroke, and/or
death has occurred, providing an opportunity for prophylactic treatment
of, for example, renal artery disease, coronary atherosclerosis, ischemic
heart disease, or peripheral artery disease (PAD).

[0020] Accordingly, in one aspect, the invention relates to a method for
determining whether blood flow is restricted in a blood vessel of an
individual suspected of compromised blood flow in the vessel, the method
comprising:

[0021] (a) delivering UTP, a derivative thereof, or a salt
thereof to said vessel,

[0022] (b) assessing blood flow quantitatively in
the vessel by obtaining a value for that indicates or correlates to blood
flow in said vessel,

[0023] (c) comparing the obtained value with a
reference value, and

[0024] (d) determining whether the individual has
compromised blood flow based on the results of the comparison.

[0025] In one embodiment of the above method, the individual is suffering
or suspected to be suffering from obesity, hypertension, vasculitis,
increased thrombotic risk, hypercholesterolemia, atherosclerosis,
diabetic complications, or vascular stenosis. In another embodiment, the
individual is suffering from Peripheral Artery Disease (PAD), coronary
atherosclerosis, atherosclerosis, renal artery stenosis, or ischemic
heart disease.

[0027] In yet another embodiment, the reference value is obtained by
measuring blood flow in another similar vessel of said individual. Blood
flow is measured, for example, by FFR, CFR, MAP, or APV measurement. In
yet another embodiment, delivery occurs by in situ infusion. Delivery can
also occur via continuous intravenous infusion, intracoronary infusion,
drip infusion, intracoronary bolus injection, guiding catheter, or an IC
microcatheter, preferably a guiding catheter or IC microcatheter.

[0028] In another aspect, the invention relates to a method for
determining whether blood flow is restricted in a blood vessel of an
individual suspected of compromised blood flow in the vessel, the method
comprising:

[0029] (a) delivering UTP, a derivative thereof, or a salt
thereof to said vessel,

[0030] (b) assessing blood flow quantitatively in
the vessel by obtaining a value that correlates to blood flow in said
vessel,

[0031] (c) comparing the obtained value with a reference value,
and

[0032] (d) determining whether the individual has compromised blood
flow based on whether there is a difference between the obtained value
and the reference value indicative of a reduction in blood flow relative
to a healthy vessel.

[0033] In yet another aspect, the invention relates to a method for
determining whether blood flow in a blood vessel of an individual
suspected of compromised blood flow in the vessel is restricted, the
method comprising:

[0034] (a) delivering UTP, a derivative thereof, or
a salt thereof to said vessel of an individual suspected of having an
atherosclerotic or ischemic disease,

[0035] (b) assessing blood flow
quantitatively in the vessel by obtaining a value that correlates to
blood flow in said vessel,

[0036] (c) comparing the obtained value with a
reference value, and

[0037] (d) determining whether the individual has
compromised blood flow based on the results of the comparison.

[0038] In yet another aspect, the invention relates to a method for
determining blood flow in a blood vessel comprising:

[0039] (a)
delivering UTP, a derivative thereof, or a salt thereof to an individual
suspected of having a vessel with compromised blood flow,

[0040] (b)
assessing blood flow quantitatively in the vessel by obtaining a value
that correlates to blood flow in said vessel,

[0041] (c) comparing the
obtained value with a reference value, and

[0042] (d) determining whether
the individual has compromised blood flow based on the results of the
comparison.

[0043] In a further aspect, the invention relates to a method for
diagnosing a disease selected from the group consisting of Peripheral
Artery Disease (PAD), coronary atherosclerosis, and ischemic heart
disease comprising:

[0044] (a) delivering UTP, a derivative thereof, or
a salt thereof to an individual suspected of having said disease,

[0045]
(b) assessing blood flow quantitatively in the vessel by obtaining a
value that correlates to blood flow in said vessel,

[0046] (c) comparing
the obtained value with a reference value, and

[0047] (d) determining
whether the individual has compromised blood flow based on the results of
the comparison.

[0048] In yet another aspect, the invention relates to a method for the
induction of maximal hyperemia comprising administering to an individual
in need thereof UTP, a derivative thereof or a salt thereof. In one
embodiment, the hyperemia is coronary hyperemia.

[0049] A further aspect of the invention relates to a kit for determining
blood flow in a blood vessel comprising: (a) UTP, a derivative thereof,
or a salt thereof as an active diagnostic ingredient, and (b)
instructions for the use thereof in an individual with suspected
compromised blood flow. In one embodiment, the kit further comprises a
microcatheter or guiding catheter. In another embodiment, the kit further
comprises a physiologically acceptable aqueous carrier, preferably
saline. In a further embodiment, the physiologically acceptable aqueous
carrier and the active diagnostic ingredient are provided in separate
containers.

[0050] In another aspect, the invention relates to a diagnostic
composition comprising UTP, a derivative thereof, or a salt thereof in a
pharmaceutically acceptable aqueous carrier suitable for administration
into a human patient, wherein the composition contains from about 50 to
about 400 μg/ml of UTP, a derivative thereof, or a salt thereof. In
one embodiment, the diagnostic composition containing an effective amount
of the diagnostic reagent is delivered to an individual in need thereof
in a total volume of about 2 ml to about 10 ml to induce hyperemia,
particularly maximal hyperemia.

[0051] In yet another aspect, the invention relates to a method for
diagnosing renal artery stenosis comprising:

[0052] (a) delivering UTP,
a derivative thereof, or a salt thereof to an individual suspected of
having said disease,

[0053] (b) assessing blood flow quantitatively in
the vessel by obtaining a value that correlates to blood flow in said
vessel,

[0054] (c) comparing the obtained value with a reference value,
and

[0055] (d) determining whether the individual has compromised blood
flow based on the results of the comparison.

[0056] In yet another aspect, the invention relates to a method for
screening individuals at risk of developing an atherosclerotic or
ischemic disease comprising:

[0057] (a) delivering UTP, a derivative
thereof, or a salt thereof to the individual,

[0058] (b) assessing blood
flow quantitatively in the vessel by obtaining a value that correlates to
blood flow in said vessel,

[0059] (c) comparing the obtained value with a
reference value, and

[0060] (d) determining whether the individual has
compromised blood flow based on the results of the comparison.

[0061] In yet another aspect, the invention relates to a method for
measuring fractional flow reserve (FFR) comprising:

[0062] (a)
delivering UTP, a derivative thereof, or a salt thereof to a blood vessel
in escalating stepwise doses,

[0064] In one embodiment, FFR measures blood flow in a coronary artery. In
another embodiment, UTP, a derivative thereof, or a salt thereof is
delivered by a delivery device, such as a microcatheter or guiding
catheter. In yet another embodiment, UTP, a derivative thereof, or a salt
thereof is delivered by intracoronary infusion. In yet a further
embodiment, the escalating stepwise doses are 20, 40, 80, 160, 240, 360,
and 400 μg/min.

BRIEF DESCRIPTION OF DRAWINGS

[0065] FIG. 1 is a graph showing mean FFR following intracoronary infusion
in a guiding catheter of UTP versus adenosine in humans with coronary
artery disease. This graph demonstrates that UTP is superior to adenosine
for lowering the FFR ratio (p=0.003). The FFR is expressed as the mean of
the 23 subjects.

[0069]FIG. 5 is a graph showing mean FFR and average peak velocity (flow)
response during micro-catheter infusion of adenosine and UTP. The graph
clearly demonstrates that at any given equipotent infusion,
UTP>adenosine and adenosine never reaches as low an FFR as UTP.

[0070] FIGS. 6A through 6D are graphs showing hemodynamic variables
(pulmonary vascular resistance, systemic vascular resistance, leg blood
flow and leg vascular conductance) during systemic UTP infusion in
comparison to adenosine, ATP, and ADP. UTP increases blood flow and leg
vascular conductance much more than the other compounds, making it
suitable for assessment of peripheral artery disease because the blood
flow increase may simulate exercise.

[0071] FIGS. 7A through 7D are graphs show hemodynamic variables (mean
arterial pressure, cardiac output, heart rate and stroke volume) during
systemic UTP infusion in comparison to adenosine, ATP and ADP. UTP does
not lower blood pressure as much as the other compounds but increases
cardiac output and HR, more resembling exercise, thus making it a
suitable stress agent for studies involving myocardial perfusion imaging.

[0072] FIG. 8 is a graph showing that use of UTP alters clinical decision
making more than adenosine because it determines the FFR more accurately.
(n=23). This would lead to a diagnostic advantage.

[0073] FIG. 9 is a diagram of indications and methods contemplated to be
within the scope of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0074] The term "hypertension", when used herein, refers to high blood
pressure. This generally means that the systolic blood pressure is
consistently over 140 and/or the diastolic blood pressure is consistently
over 90. Hypertension is when either or both of the systolic blood
pressure and the diastolic blood pressure are too high.

[0075] "Ischemic heart disease" or myocardial ischemia as used herein
refers to a disease characterized by reduced blood supply to the heart
muscle, usually due to coronary artery disease (atherosclerosis of the
coronary arteries).

[0076] A "stenosis" as used herein, is be defined as an abnormal narrowing
in a blood vessel or other tubular organ or structure.

[0077] "Individual" or "subject" as used herein is intended to mean any
mammal, including a human, veterinary animal, (such as a farm animal, a
domestic animal), or laboratory animal (such as a rodent or a primate).

[0078] "P2Y2 receptor" as used herein is intended to mean a G
protein-coupled extracellular nucleotide receptor associated with a PI
signalling pathway, which may be activated for example by extracellular
nucleotides.

[0079] "Normal blood flow" as used herein is intended to mean blood flow
that is uncompromised by, for example, a stenosis or a blood clot. A
standard for normal blood flow may for example be developed by measuring
the blood flow in at least 20 healthy individuals with no suspected
illnesses and determining the average.

[0080] "Compromised blood flow" as used herein is intended to mean any
abnormality in which blood flow through a vessel is lower than normal
blood flow due to a constriction, or mechanical obstruction or
inflexibility in the vessel wall, such as stenosis. Compromised blood
flow may be assessed by many parameters including but not limited to a
drop in blood pressure and may be measured in arteries and veins as well
as in functional tissue.

[0081] "Hyperemia" as used herein is intended to mean the increase of
blood flow to different tissues in the body.

[0082] "Maximal hyperemia" as used herein is intended to mean maximal
increase in blood flow, which can be induced by the administration of
modulators of the P2Y2 receptor, which according to the present invention
is UTP, derivatives thereof, or salts thereof as further defined below.
This can be measured, inter alia, by measuring the pressure difference
between the proximal end and the distal end of a blood vessel suspected
of compromised blood flow. When using this methodology, maximal hyperemia
is considered reached when upon further supply of UTP, a UTP derivative,
or a salt thereof, the distal pressure does not change.

[0083] "Ankle-brachial index" as used herein is intended to mean the ratio
of the blood pressure in the lower legs to the blood pressure in the arms

[0084] "Derivative" as used herein is intended to refer to substitution(s)
or modification(s) of any group or groups on UTP, not limited to those
disclosed herein, that results in a compound that activates the P2Y2
receptor to a degree in which the ratio of the EC50 for stimulation of
the human P2Y2 receptor divided by the EC50 for stimulation of the human
P2X1 receptor is higher, such as at least 2 fold higher, than the
corresponding ratio for ATP. Examples of such derivatives include, but
not limited to, UTP.sub.γS, MRS2498, uridine 5'-trisphosphate tris
salt, uridine 5'-trisphosphate salt dihydrate, uridine 5'-trisphosphate
salt solution, uridine 5'-trisphosphate salt hydrate,
uridine-13C9, 15N2 5'-trisphosphate sodium salt
solution, uridine-15N2 5'-trisphosphate sodium salt solution,
uridine 5'-triphosphate trisodium salt hydrate, uridine-13C9,
15N2 5'-triphosphate sodium salt solution,
uridine-15N2 5'-triphosphate sodium salt solution, 2-diuridine
tetraphosphate, thioUTP tetrasodium salt, denufosol tetrasodium, or
UTP.sub.γS trisodium salt.

[0085] The term "significant difference" is used herein to mean that the
difference in a measured value (e.g., in amount of blood flow determined
in a subject) and a reference value is indicative of restricted blood
flow.

[0086] The term "reference value" may have different meanings depending on
context. For example, in some cases, a "reference value" refers to the
range of normal values for blood flow (which may be assessed directly or
indirectly by measuring another variable that correlates to blood flow).
Alternatively, a "reference value" may represent the value of blood flow
associated with an abnormal condition. For example, when fractional flow
reserve is used to assess blood flow, the measured value is pressure
differential between the distal end of a stenotic blood vessel segment
under conditions of maximal hyperemia and the reference value is the
corresponding pressure differential under similarly hyperemic conditions
in the same vessel without stenosis. The ratio of the two provides the
comparison. If the ratio is 1 (and blood is actually flowing through the
vessel) there is no stenosis. It is also possible, however, for a
reference value to be a value indicating abnormality (usually a threshold
value), in which case, the comparison would show whether the measured
value has a certain relationship to the reference value (e.g., higher or
lower than the threshold abnormality value) The connotation of "reference
value" as used in a specific context will be apparent to one of ordinary
skill in the art.

[0087] "About" as used herein is intended to mean within a statistically
meaningful range of a value. Such a range can be within an order of
magnitude, preferably within 50%, more preferably within 20%, more
preferably still within 10%, and even more preferably within 5% of a
given value or range. The allowable variation encompassed by the term
"about" depends on the particular context under study, and can be readily
appreciated by one of ordinary skill in the art. For example, "about" may
reflect experimental error or experimental variation in measurement or
interpatient differences. As a specific example, "about 20 seconds" when
referring to terminal half-life of UTP would encompass inter-patient
differences of up to 10 to 60 seconds depending upon ectonucleotidase
activity in a given patient.

[0088] "Delivery" as used herein is intended to include any administration
that causes an effective concentration of the administered substance to
be in contact with the vessel being assessed. Included without limitation
are in situ administration to the vessel, for example, infusion using a
guiding, FFR/thermodilution catheter (PressureWire® Certus (St. Jude
Medical, Inc.)) which enables simultaneous measurement of thermo-dilution
flow (via infusion of saline) and concurrent gathering of FFR and CFR
(coronary flow reserve), or a microinfusion catheter, or systemic
administration to the bloodstream via an intravenous catheter.

[0089] "At risk" as used herein is intended to refer to individuals with a
genetic predisposition to developing a vascular disease or individuals
who have undergone a procedure to treat a vascular disease. A genetic
predisposition may, for example, be a mutation in a gene required for
normal organ function. Individuals who have undergone a procedure to
treat a disease may be at risk for redeveloping the disease or condition,
for example, restenosis.

[0090] "Fractional flow reserve" or "FFR" as used herein is intended to
mean the ratio of absolute distal pressure to proximal pressure at
maximal hyperemia. FFR is defined as an index described as the ratio of
the hyperemic flow in a stenotic artery to the hyperemic flow in the same
artery if there was no stenosis present.

EMBODIMENTS

[0091] The methods described herein are useful in determining compromised
blood flow in an individual with suspected compromised blood flow. Such
methods mimic the increased blood flow that occurs during exercise and
are thus particularly useful in patients who cannot undertake exercise or
for whom it is less desirable to undertake exercise.

[0092] The present invention relates to a method for determining whether
blood flow is restricted in a blood vessel of an individual suspected of
compromised blood flow in the vessel, the method comprising the steps of:
(a) delivering UTP, a derivative thereof, or a salt thereof to said
vessel, (b) assessing blood flow quantitatively in the vessel by
obtaining a value that indicates or correlates to blood flow in said
vessel, (c) comparing the obtained value with a reference value, and (d)
whether the individual has compromised blood flow based on the results of
the comparison.

[0093] The methods of the invention may be used to determine a suspected
compromised blood flow in any blood vessel in the body. Thus, UTP, a
derivative thereof, or a salt thereof, can be delivered to any blood
vessel in the body. While the vessel is preferably an artery, it is also
possible to determine compromised blood flow in a vein. Compromised blood
flow in a vein may, for example, be caused by a stenosis in a vein, such
as a stenosis in a graft from a bypass surgery. Within the scope of the
present invention is included measuring the blood flow across any artery.
In preferred embodiments, blood flow is measured in a coronary artery,
such as the main stem artery, right coronary artery, left coronary
artery, or any other appropriate coronary artery. In other equally
preferred embodiments, blood flow is measured in the any of the common
iliac arteries, including, but not limited to, the femoral artery, iliac
artery, or popliteal artery.

[0094] By delivering UTP, a derivative thereof, or a salt thereof, in
accordance with the method of the invention, hemodynamic conditions can
be diagnosed more effectively, with reduced or eliminated side-effects,
thereby allowing the physician to determine the extent of abnormality, if
any, more accurately, and make a more informed choice as to the course of
treatment, if any, to be pursued.

[0095] For instance, atherosclerosis is a condition in which an artery
wall thickens as the result of a buildup of fatty materials such as
cholesterol. Atherosclerosis may give rise to various symptoms (e.g.,
claudication, angina pectoris), and depending on the symptoms,
atherosclerosis may be referred to as, for example, PAD (peripheral
artery disease), coronary atherosclerosis, or TCI (transient coronary
ischemia).

[0096] Using the methods described herein, it is possible to determine
compromised blood flow, which may be an indication that an individual is
suffering from an atherosclerotic disease. The atherosclerotic disease
may be any atherosclerotic disease, for example, coronary artery disease
(CAD), Peripheral Artery Disease (PAD), renal artery disease, vascular
stenosis, aortic stenosis, renal artery stenosis, and coronary
atherosclerosis. In a particular embodiment, the disease is CAD.

[0097] Coronary artery disease and its clinical manifestations, such as
myocardial infarction, are heritable traits. While methods such as
percutaneous coronary intervention (PCI) can successfully treat coronary
artery disease, there still remains a need for effective diagnostic
screening methods. Such diagnostic screening methods are important not
only in those afflicted with the disease, but also to screen those at
risk for developing the disease due to, for example, restenosis or a
genetic predisposition to coronary artery disease. Such screening methods
can be, but are not limited to, FFR, CFR, MAP, APV measurements. Thus,
methods of the present invention can be applied to screen at-risk
patients. These screening methods need not be limited only to at-risk
individuals, but can, for example, be incorporated into routine heath
checkups and performed on a regular basis.

[0098] Coronary artery disease and its clinical manifestations, including
myocardial infarction, are heritable traits, consistent with a role for
inherited DNA sequence variation in conferring risk for disease. There
are many modifiable risk factors for heart disease, such as smoking or
exposure to environmental tobacco smoke, obesity, sedentary lifestyle,
diabetes, high cholesterol or abnormal blood lipids, and hypertension.
There are also non-modifiable risk factors, such as male sex, age>50
years, and a family history of heart disease. Many biological markers,
including elevated levels of homocysteine, are associated with an
increased risk for atherosclerosis and it has been recognized that some
people have a common defective genetic variant (called
methylenetetrahydrofolate reductase, "MTHFR") that leads to elevated
levels of homocysteine. Furthermore, certain genes are associated with
increased risk of CAD. One example is a common polymorphism located on
chromosome 9p21.3. Moreover, many loci, including 9p21, are located in
intergenic segments and elicit the phenotype by novel mechanisms which
need further elucidation.

[0099] Methods of the invention can also be used to determine compromised
blood flow in an individual who is suffering or suspected of suffering
from atherosclerosis, obesity, hypertension, vasculitis, increased
thrombotic risk, hypercholesterolemia, diabetic complications, or
vascular stenosis. Compromised blood flow may also be an indication that
an individual is suffering from ischemic heart disease. Ischemic or
ischemic heart disease (IHD), or myocardial ischemia, is a disease
characterized by reduced blood supply to the heart muscle, usually due to
coronary artery disease (atherosclerosis of the coronary arteries).

[0100] The methods described herein are also useful for the determination
of compromised blood flow, which may also be an indication that an
individual has a blood clot. A blood clot or a thrombus is the
inappropriate activation of the hemostatic process in an uninjured or
slightly injured vessel. A thrombus in a large blood vessel (mural
thrombus) will decrease blood flow through that vessel. In a small blood
vessel (occlusive thrombus), blood flow may be completely cut-off,
resulting in death of tissue supplied by that vessel. Thus, in one
embodiment of the invention, the suspected compromised blood flow is
caused by stenosis, particularly a coronary stenosis or any stenosis in
the iliacs, such as femoral arterial stenosis.

[0101] PAD is an atherosclerotic disease that leads to a narrowing of the
arteries, particularly in the legs. This narrowing (i.e., stenosis)
limits the amount of blood able to pass through the arteries, resulting
in claudication. PAD is associated with significant morbidity and
mortality. Medical therapy, including risk factor modification and
antiplatelet medications, reduces cardiovascular morbidity and mortality
rates in patients with PAD. This is why availability of safe, effective
and improved diagnostics is crucial.

[0102] Patients who cannot perform treadmill exercises are presently
tested with active pedal plantar flexion or with inflation of a thigh
cuff well above systolic pressure, in an attempt to produce "reactive"
hyperemia. Unfortunately, many patients do not tolerate the discomfort
associated with this degree and duration of cuff inflation, so this
method is rarely performed. Therefore, pharmacological diagnostics
methods which selectively increase blood flow to the legs at rest and
simulate exercise are desirable alternatives. UTP will, when infused
systemically (i.v.), (Example 3), induce increases in cardiac output by
increasing heart rate (thus simulating exercise), thereby increasing
blood flow to the legs. This makes a stenotic lesion easily detectable by
methods for assessing blood flow. Doppler ultrasound, ankle brachial
index monitoring, and the like, as described herein. Thus, in one
embodiment, UTP, a derivative thereof, or a salt thereof, is contemplated
for use in determining whether blood flow is restricted in a blood vessel
of an individual suffering or suspected of suffering from PAD.

[0103] Renal artery stenosis often leads to drug resistant hypertension.
Determination of renal arterial stenosis severity can be assessed in a
similar manner as coronary artery stenosis by use of the pressure
gradient and vessel diameter in the kidney [24]. Renal arteries can be
examined bilaterally using the same femoral approach as coronary FFR, and
bilateral selective renal arteriograms can be obtained. By utilizing the
vasoactive effects of UTP to induce renal hyperemia, a pressure gradient
across the renal arteries can be assessed. While adenosine lowers
glomerular filtration rate by constricting afferent arterioles and causes
dose-dependent renal vasoconstriction [25], UTP induces renal
vasodilatation [26]. Therefore, a combined catheter with pressure and UTP
infusion ensures local infusion and avoids systemic spill over. Thus,
UTP, a derivative thereof, or a salt thereof, can be used in conjunction
with the methods described herein to detect the presence of renal
stenosis.

[0104] UTP, a derivative thereof, or a salt thereof, can also be used for
the noninvasive testing for renal artery stenosis by so-called Duplex
scanning, which is a non-invasive ultrasound method that is both
sensitive and specific for detecting stenotic lesions and the severity of
the stenosis. They can then be categorized and UTPs hemodynamic
significance or renal blood flow can be evaluated. Noninvasive diagnostic
technologies continue to advance, and as new methods are validated, the
need for renal arteriography may lessen. UTP infusion during duplex scan
could be such a diagnostic test.

[0105] Stenosis can have many causes. A stenosis within the scope of the
present invention may have any underlying cause including, but not
limited to, stenosis caused by atherosclerosis, ischemia, infection,
neoplasm, inflammation, or smoking. Thus, in a particular embodiment, the
methods described herein may be used for the determination of compromised
blood flow, which may be an indication that an individual has a stenosis.

[0106] Methods described herein can also be applied to the detection of
hyperproliferative vascular diseases resulting from mechanical injury,
for example, that arising from the use of stents, catheters, and the
like.

UTP, UTP Derivatives, and UTP Salts

[0107] UTP is available from commercial sources (e.g., Sigma Aldrich (St.
Louis, Mo.), Trilink Biotechnologies, Inc. (San Diego, Calif.), Axxora
(Nottingham, England and Loerrach, Germany), Torcis Bioscience
(Ellisville, Mo.), Inspire Pharmaceuticals, Inc. (Durham N.C.)).
Nucleoside phosphates are also commercially available (Sigma Aldrich) or
can be made from the corresponding nucleosides by methods known to those
skilled in the art. Likewise, where nucleosides are not commercially
available, they can be made by modifying readily available nucleosides,
or by synthesis from heterocyclic and carbohydrate precursors by methods
known to those skilled in the art.

[0108] UTP, a derivative thereof, or a salt thereof, used in the invention
is capable of stimulating the human P2Y2 receptor to a degree in which
the ratio of the EC50 for stimulation of the human P2Y2 receptor divided
by the EC50 for stimulation of the human P2X1 receptor is higher, such as
at least 2 fold higher, than the corresponding ratio for ATP.

[0109] Without being bound a specific theory, it is believed that any
UTP-related compound that has such a higher ratio as compared to ATP
provide the same advantages as ATP in that they can increase blood flow
and override increases in muscle sympathetic vasoconstrictor activity,
but do not have, or have to a lesser degree, the disadvantages of ATP,
i.e., the activation of purinergic P2X receptors, which results in
vasoconstriction and risk of hypertension.

[0110] The formula for UTP and certain derivatives is provided below.

[0111] The formula for UTP and certain derivatives is provided below.

##STR00001## ##STR00002## ##STR00003##

[0112] A UTP derivative has a modification or substitution of one or more
residues of UTP. Preferably, a UTP derivative is a compound comprising
UTP, wherein one or more --H are exchanged for another group, such as one
or more --H groups of the ribose moiety or one or more --H groups of the
pyrimidine moiety. Preferably said --H is exchanged with another group
selected from the group consisting of lower alkyl, lower alkenyl, lower
alkoxy, lower alcohol, --OH, lower amines, --NH2 and halogen. Lower
in this sense means C1-6, preferably C1-3, and thus by way of
example a lower amine, may for example be C1-6-alkyl-NH2 or a
C1-6-alkenyl-NH2. Examples of UTP derivatives include, but are
not limited to, 5-substituted UTP-derivatives, for example 5, alkyl
substitutions, and C'-alkyl UTP derivatives, for example, containing
alkyl groups in different positions of the ribose moiety. Alkyl
substitutions include, but are not limited to, methyl, ethyl, proplyl,
pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl and dodecyl
substitutions [27].

[0113] Other substitutions include, but are not limited to, methylene,
propylene, amino, sugar, any halogen and propenyl substitutions at any
uridine residue. A UTP derivative may have one or more modifications
and/or substitutions. For example, a UTP derivative may have one
modification and/or substitution, such as two modifications and/or
substitutions, for example three modifications and/or substitutions or
four modifications and/or substitutions.

[0117] In other particular embodiments. the compound used in the invention
is a compound of the general formula (I):

##STR00004##

wherein, R1 is O, S, hydroxyl, mercapto, amino or cyano, R2 is H, Br,
nothing, acyl, C1-6 alkyl or sulphonate, R3 is O, S, hydroxyl,
mercapto or amino, R4 is H, hydroxyl, methyl, cyano, nitro, halogen such
as Br, R5 is H or Br X1, X2, X3 and X4 are
independently 0'' or S'', Y is O, imido, methylene or dihalomethylene,
such as difluoromethylene, Z is CH2, n and m are independently 0 or
1, and n+m is 0, 1 or 2, A is H or ribose, linked at the 5 position with
a pyrimidine or purine residue or pyrimidine or purine derivative
selected from the group of uracil, cytosine, guanine, adenine, xanthine,
hypoxanthine, linked through the 1 or 9 position, respectively or ribose
linked at the 5 position with a pyrimidine residue having formula II

##STR00005##

wherein the denoted R groups are as listed above or ribose lined at the 5
position with a purine residue having formula (III)

##STR00006##

wherein R6 is NH2, while R7 is nothing and there is a double bond between
N1 and C6 (adenine) or wherein R6 is NH2 and R7 is 0 and there is a
double bound between N1 and C6 (adenine-1-oxide) or wherein R6 and R7
together form a a ring of --NCH═CH-- from N6 to N1 with a double
bound between N-6 and C6 (1, N6-ethenoadenine),

[0118] In a further embodiment, the compound used is P1-(uridine
5')--P4-(2'-deoxycytidine 5')tetraphosphate or a salt thereof, such
as a tetrasodium salt (INS37217).

[0119] In yet further embodiments, the compound capable of stimulating the
P2Y2 receptor is one of the compounds described in U.S. Pat. No.
5,292,498 (paragraph 2, line 1 to line 32) and U.S. Pat. No. 5,789,391
(paragraph 2, line 40 to paragraph 3, line 55). In yet another
embodiment, the compound used is a P2Y2 agonist described in U.S. Pat.
No. 5,837,861, such as P1,P4-Di(uridine 5'-P2,P3-methylene
tetraphosphate), P1,P4-Di(uridine
5'-P2,P3-difluoromethylenetetraphosphate), P1.P4-Di(uridune
5'-P2,P3-imidotetraphosphate), P1,P4-Di(4-thioruridine
5'-tetraphosphate), P1,P5-Di(uridine 5'-pentaphosphate), and
P1,P4-Di(3,N4-ethenocytidine 5'-tetraphosphate).

[0120] UTP, a derivative thereof, or a salt thereof, may be formulated as
a free base or salt. Pharmaceutically acceptable salts include acid
addition salts (formed with the free amino groups of the peptide
compound) and which are formed with inorganic acids such as, for example,
hydrochloric or phosphoric acids, or such organic acids as acetic acid,
oxalic acid, tartaric acid, mandelic acid, and the like. Salts formed
with the free carboxyl group may also be derived from inorganic bases
such as, for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine, trimethylamine,
2-ethylamino ethanol, histidine, procaine, and the like.

[0121] Preferred salts of UTP, a derivative thereof, or a salt thereof,
are alkali salts or alkali earth salts, such as sodium salts, potassium
salts, calcium salts and magnesium salts. Other preferred salts include,
but are not limited, to tris salts, hydrates and dihydrates. The UTP salt
may comprise one or more of the above mentioned salts on any UTP residue,
such as disalts, trisalts and tetrasalts, for example disodium salts,
dipotassium salts, dicalcium salts and dimagnesium salts, as well as
trisodium salts, tripotassium salts, tricalcium salts and trimagnesium
salts and tetrasodium salts, tetrapotassium salts, tetracalcium salts and
tetramagnesium salts. The UTP salts may be substituted on any UTP
residue, preferably the salts are 5' or C' substituted.

Formulations and Modes of Administration

[0122] UTP, a derivative thereof, or a salt thereof may be delivered in
any suitable way known in the art. Preferred modes of delivery include
parenteral, intravenous, intra-arterial, in situ infusion, and the like.

[0123] UTP, a derivative thereof or a salt thereof may be formulated for
parenteral administration (e.g., by injection, for example bolus
injection or continuous infusion) and may be presented in unit dose form
in ampoules, pre-filled syringes, small volume infusion or in multi-dose
containers with an added preservative. A diagnostic composition for
parenteral administration may include sterile aqueous and non-aqueous
injectable solutions, dispersions, suspensions or emulsions in oily or
aqueous vehicles, for example solutions in aqueous polyethylene glycol,
as well as sterile powders or lyophilisates to be reconstituted in
sterile injectable solutions or dispersions prior to use.

[0124] UTP, a derivative thereof, or a salt thereof, may be in powder
form, obtained by aseptic isolation of sterile solid or by lyophilisation
from solution for constitution before use with a suitable vehicle, e.g.,
sterile, pyrogen-free water. Aqueous solutions should be suitably
buffered if necessary, and the liquid diluent first rendered isotonic
(i.e., to an osmolarity of about 300 mOsm) with sufficient saline or
glucose. Solubility of UTP, a derivative thereof, or a salt thereof
increases by warming and lowering the pH of the aqueous solution. The
resulting aqueous solution can be sterilized by filtration. The aqueous
solutions can also be heated to a sterilization temperature, e.g.,
99° C. for 10 min at physiological pH values, e.g., in order to
inactivate enzymes such as ectonucleotidases and ectophosphatases,
without degradation of the nucleotides. The aqueous solutions are
particularly suitable for in situ infusion, and intravenous,
intramuscular, subcutaneous and intraperitoneal delivery. The sterile
aqueous media employed are all readily available by standard techniques
known to those skilled in the art. The reagent and vehicle, of course,
can be provided in ready-to-use (pre-sterilized) form after
reconstitution at the use point.

[0125] Solutions of compounds or pharmaceutically acceptable salts thereof
can be prepared in water or saline, and optionally mixed with a nontoxic
surfactant. Compositions for in situ infusion, or intravenous or
intra-arterial delivery may include sterile aqueous solutions that may
also contain buffers, liposomes, diluents and other suitable additives.

[0126] The compounds of the present invention may be administered
parenterally in a sterile medium. The compound, depending on the vehicle
and concentration used, can either be suspended or dissolved in the
vehicle. Advantageously, adjuvants such as local anesthetics,
preservatives and buffering agents can be dissolved in the vehicle
including UTP degradation enzyme blockers. The sterile injectable
preparation may be a sterile injectable solution or suspension in a
non-toxic parentally acceptable diluents or solvent. Among the acceptable
vehicles and solvents that may be employed are physiological saline,
sterile water or Ringer's solution.

[0127] The parenteral compositions can be presented in unit-dose or
multi-dose sealed containers, such as ampules and vials, and can be
stored in a freeze-dried (lyophilized) condition requiring only the
addition of the sterile liquid excipient, for example, water, for
injections, immediately prior to use. Extemporaneous injection solutions
and suspensions can be prepared from sterile powders, granules, and
tablets of the kind previously described.

[0128] The dosage forms suitable for injection or infusion can include
sterile aqueous solutions comprising the active ingredient. In all cases,
the ultimate dosage form must be sterile, fluid and stable under the
conditions of manufacture and storage.

[0129] Sterile injectable solutions can be prepared by incorporating the
compound(s) or pharmaceutically acceptable salt(s) thereof in the
required amount in the appropriate solvent with various of the other
ingredients enumerated above, as required, followed by filter
sterilization.

[0130] In preferred embodiments, UTP, a derivative thereof or a salt
thereof, is formulated in liquid form for delivery via continuous
intravenous infusion, in situ infusion, intracoronary infusion, drip
infusion, intracoronary bolus injection, a guiding catheter, or an IC
micro-catheter. In other preferred embodiments, UTP, a derivative
thereof, or a salt thereof, is formulated in liquid form for
administration intravenously in legs for the determination of blood flow
in the legs. UTP, a derivative thereof, or a salt thereof, can also be
formulated in liquid form for delivery to the kidneys or via
intracoronary infusion to the heart for the determination of blood flow.

[0131] Other suitable embodiments relate to a diagnostic composition
comprising UTP, a derivative thereof, or a salt thereof, in a
pharmaceutically acceptable aqueous carrier suitable for administration
into a human patient, said composition containing said compound in the
range of 10 to 1000 μmol/ml, preferably in the range of 50 to 400
μmol/ml, more preferably in the range of 100 to 360 μmol/ml.
Examples of such compositions include:

Composition I: The concentration of UTP, a derivative thereof, or a salt
thereof in the injectable composition can be between 10-2000 μg/ml,
with a preferred diagnostic concentration between 20-500 μg/ml for IC
use and 50-2000 μg/ml for iv use. Composition II: The concentration of
UTP, a derivative thereof, or a salt thereof can be controlled by adding
sterile aqueous solution to dry powder UTPtrissalt or UTPNa3 by
techniques know to those skilled in the art to bring the concentration to
about 100 μg/ml for IC and 300 μg/ml for iv use, the limit of its
solubility under ambient conditions. Composition 3: The concentration of
UTP, a derivative thereof, or a salt thereof can be, in a clinical dose
range, 10-1000 μg/ml, with preferred ranges of 25-500 μg/ml, 20-100
μg/ml, 40-150 μg/ml, 60-160 μg/ml, 80-360 μg/ml, 100-240
μg/ml, 120-480 μg/ml, and 150-600 μg/ml.

[0132] A related embodiment relates to a diagnostic composition wherein
UTP, a derivative thereof, or a salt thereof, is in isotonic saline.

[0133] The rate of intravascular infusion of UTP, a derivative thereof, or
a salt thereof, in increasing order of preference, is about 20 μg to
about 2000 μg per minute, about 50 μg to about 600 μg, about 80
μg to about 360 μg, about 100 μg to about 500 μg, about 150
μg to about 400 μg, about 180 μg to about 360 μg, about 240
μg to about 360 μg, and about 180 μg to about 240 μg per
minute.

[0134] UTP can be delivered by intracoronary infusion continually or by
bolus via a guiding, FFR/thermodilution catheter or microinfusion
catheter by stepwise dose escalation starting at 20 μg/min to induce
maximal coronary blood flow, which corresponds to minimal distal coronary
pressure. When steady-state hyperemia is achieved, preferably with
continuous infusion (i.e., no further decrease in Pd is occurring),
FFR can be calculated as the ratio of the mean distal intracoronary
pressure measured by the pressure wire to the mean arterial pressure
measured by the coronary catheter. As such, a stepwise dose escalation
from 20 μg/min to about 400 μg/min during continuous UTP infusion
in any given assessed coronary artery should render the lowest possible
Pd and therefore the most accurate FFR value. Thus, hyperemic
stimuli can be given as follows: an IC continuous infusion of UTP, a
derivative thereof, or a salt thereof in incremental doses of 10, 20, 40,
80, 160, 240, 360 and 400 μg/min, in both the left and right coronary
artery depending on lesion anatomy.

[0135] When UTP, a derivative thereof, or a salt thereof is administered
via continuous intracoronary infusion, the rate of infusion maybe about 5
μg to about 600 μg/min, about 10 μg to about 550 μg/min,
about 20 μg to about 500 μg/min, about 30 μg to about 450
μg/min, about 50 μg to about 400 μg/min, about 60 μg to about
360 μg/min, about 80 μg to about 360 μg/min, and about 180 μg
to about 360 μg/min.

[0136] In terms of the solution to be infused, the UTP concentration can
vary. Typically, the concentration will be from about 50 to about 100
μg/ml to be administered at a rate of 1-5 ml/min for about 3 to 5
minutes. Thus, a vial of pre-made solution of UTP should conveniently
contain at least 10 ml of solution and could contain up to 25 ml. Larger
amounts are possible but unnecessary for individual use.

[0137] If UTP is provided in solid form (e.g., lyophilized) in a vial it
can be provided in individual use amounts according to the foregoing
guidelines or in bulk and then dissolved prior to use in an appropriate
amount of aqueous solvent.

[0138] The amounts given herein apply, on a UTP basis, to UTP salts and
derivatives, but may need to be adjusted to account for differences in
potency.

[0139] A typical practice is as follows: UTP solutions are freshly
prepared from sterile lyophilized powder (2-mL ampoules containing 20 mg
UTP as tris salt or trisodium salt) and then diluted appropriately in
aqueous NaCl 0.9%. The solution can then be passed through a 0.2-microm
Millipore filter is in a concentration of 50 microgram/ml which can then
be pushed by an infusion rate of 1-5 ml/min in order to give a
concentration of 50 to 250 microgram/min. If higher concentrations are
needed to produce an even lower FFR the infusion rate can be increased
even further, if for instance the patient has some of the known P2Y2
receptor polymorphisms (see, e.g., Janssens, R. et al, "Human P2Y2
receptor polymorphism: identification and pharmacological
characterization of two allelic variants" Br J. Pharmacol. 1999 June;
127(3):709-16; and Buescher, R. et al, "P2Y2 receptor polymorphisms and
haplotypes in cystic fibrosis and their impact on Ca2+
influx"Pharmacogenet Genomics. 2006 March; 16(3):199-205).

[0140] The drug may be administered at any time when the patient is in
need of a hyperemic assessment such as FFR or other related diagnostic
procedures, such as another form of nuclear imaging (including MPI),
Ultrasound and echo cardiography, Fractional flow reserve, MRI/MRA, CT
scan, PET scan, and ankle brachial index using mean arterial pressure.

Methods for Assessing Compromised Blood Flow

[0141] Methods of assessing compromised blood flow include nuclear imaging
such as MPI, ultrasound and echo cardiography, fractional flow reserve,
MRI/MRA, CT scan, PET scan, and ankle brachial index using mean arterial
pressure. Compromised blood flow means any alteration in blood flow
compared to normal blood flow, for example, an alteration caused by a
stenosis or blood clot. Conversely, normal blood flow means blood flow
that is uncompromised by, for example, a stenosis or a blood clot. A
reference for normal blood flow may, for example, be developed by
measuring the blood flow in at least 20 individuals with no suspected
illnesses and determining the average. Conversely, some measurements are
specific to the individual, e.g., FFR measurements. That is, the FFR
value is solely dependent on the individual's ability to increase blood
flow compared to when at rest.

[0142] In other contexts, a "reference value" may represent the value of
blood flow associated with an abnormal condition. For example, when FFR
is used to assess blood flow, the measured value is pressure differential
between the distal end of a stenotic blood vessel segment under
conditions of maximal hyperemia and the reference value is the
corresponding pressure differential under similarly hyperemic conditions
in the same vessel without stenosis. The connotation of "reference value"
as used in a specific context will be apparent to one of ordinary skill
in the art.

[0143] To determine whether a blood vessel has compromised blood flow, the
blood flow can be assessed quantitatively by obtaining a value that
correlates to blood flow in the vessel. This obtained value can then be
compared to a reference value, which can be obtained, for example, from a
similar blood vessel in the individual. It can then be determined whether
the individual has a compromised blood flow based on the results of the
comparison. This comparison allows for the determination of compromised
blood flow in a blood vessel and may thereby assist in determining the
presence of a stenosis and/or in diagnosing a disease or disorder, for
example, Peripheral Artery Disease (PAD), coronary atherosclerosis, renal
artery disease, atherosclerosis and/or ischemic heart disease.

[0144] The similar blood vessel may be any vessel with the same cross
section area+/-20%. The similar vessel may thus, for example, be a
similar blood vessel in the same individual, such as a vessel in same
individual with the same cross section area+/-20%, and which has the same
distance to the heart+/-20%. For example, if the blood vessel with
suspected compromised blood flow is on the left side of the body, the
similar vessel may be the corresponding vessel on the right side of the
body. The similar vessel can, however, also be the same vessel in another
healthy individual.

[0145] When blood flow is measured in the legs, the similar blood vessel
with normal blood flow may be the vessel in one leg of an individual,
wherein the vessel with a suspected compromised blood flow is in the
other leg. When blood is measured in the heart, the similar blood vessel
with normal blood flow may be one coronary artery in an individual,
wherein the blood vessel with a suspected compromised blood flow is in
another coronary artery.

[0147] FFR was originally used in coronary catheterization to measure
pressure differences across a coronary artery stenosis to determine the
likelihood that the stenosis impedes oxygen delivery to the heart muscle,
and is defined as the pressure behind (distal to) a putative stenosis
relative to the pressure before the putative stenosis. The result is an
absolute number; an FFR of 0.50 means that a given stenosis causes a 50%
drop in blood pressure across the stenotic area. That is, FFR expresses
the maximal flow down a vessel in the presence of a stenosis compared to
the maximal flow in the hypothetical absence of the stenosis. During
coronary catheterization, a catheter is inserted into the femoral (groin)
or radial arteries (wrist) using a sheath and guidewire. FFR uses a small
sensor on the tip of the wire (commonly a transducer) to measure
pressure, temperature and flow to determine the exact severity of the
lesion. This is done during maximal blood flow (hyperemia). A pullback of
the pressure wire is performed, and pressures are recorded across the
vessel.

[0148] FFR measurements can be carried out in any blood vessel in the
body, for example, those in the legs, kidney, or heart. Any variation to
the FFR method is contemplated to be within the scope of the present
invention. In specific embodiments, the FFR method uses a guidewire or
the tip of an IC microcatheter (Progreat Microcatheter System, Terumo,
Japan).

[0149] Other catheters suitable for use in conjunction with the invention
are disclosed in U.S. Provisional application Ser. No. 61/357,857, which
is hereby incorporated by reference.

[0150] Advantages of using UTP over adenosine compounds in FFR include:
the instantaneous achievement of steady state by UTP, making it feasible
to perform accurate measurements and the pullback maneuver and bringing
FFR procedure time down; the more accurate estimation of coronary blood
flow, resulting in a more precise FFR because UTP produces close to
maximum perfusion; UTP is not associated with side effects; UTP allows
for a clear dose response curve, and UTP is short-acting, with no
long-term effects. There is also emphasis on procedure-related
complications and this new method therefore allows for repeated and easy
measurement of FFR and can be performed via the radial artery on an
outpatient basis, whereas intravenous adenosine will always require
central venous access.

[0151] MAP is the perfusion pressure seen by organs in the body.
Compromised blood flow may, in the order of preference, be reflected in a
MAP of less than 60 mmHg, less than 50 mmHg, less than 40 mmHg, less than
30 mmHg, or less than 20 mmHg.

[0152] Average peak velocity (APV) may also be used in the present
invention to determine the suspected compromised blood flow. APV in the
range of 5 to 30 msec-1 is indicative of compromised blood flow.
Thus, in order of preference, APV of less than 30 msec-1, preferably
less than 20 msec-1, and more preferably less than 10 msec-1
are considered indicative of compromised blood flow.

[0154] Contraindications to dobutamine include: chest pain, high blood
pressure, dizziness, nausea and extreme fatigue, ingestion of caffeine
and treatment with beta blockers. Given that current risks of the stress
echocardiogram procedure with dobutamine are not seen with UTP,
dobutamine can be substituted with UTP, a derivative thereof, or a salt
thereof in stress echocardiograms to assess blood flow in an blood
vessel, to assess the heart's function and structures such as valve
stenosis, to determine limits for safe exercise in patients who are
entering a cardiac rehabilitation program and/or those who are recovering
from a cardiac event, such as a heart attack (myocardial infarction, or
MI) or heart surgery, to evaluate blood pressure during stress testing,
to assess stress or exercise tolerance in patients with known or
suspected coronary artery disease or to evaluate the cardiac status of a
patient about to undergo surgery with for instance aortic stenosis.

[0155] MPI is the most widely used non-invasive method used for the
detection of coronary artery disease, risk assessment, detection of
viable myocardium, and evaluation of the effects of various therapeutic
interventions. Adenosine and dipyridamole have been the mainstays of
vasodilator stress testing. Dobutamine stress MPI is reserved for
patients with contraindications for vasodilator testing with adenosine.
However, all adenosine receptor agonists, including regadenoson, have
been associated with bronchoconstriction, angina, severe hypotension and
sinoatrial and atrioventricular nodal block because adenosine receptor
agonists can depress the SA and AV nodes and may thus cause first-,
second- or third-degree AV block, or sinus bradycardia; thus the drugs
should not be given to patients with 2nd or 3rd degree AV block, or sinus
node dysfunction. Importantly, UTP has none of these disadvantages, being
more receptor selective and closer to maximal coronary blood flow and as
there are no "adenine" effects of the current compound as it acts through
a completely different purinergic receptor system (P2Y2). It is therefore
much safer to use systemically because of limitation to systolic and
diastolic blood pressure drops+no effect on the AV node. As such, UTP, a
derivative thereof, or a salt thereof can be used in place of adenosine
receptor agonists in MPI.

Methods for Detecting Blood Flow

[0156] Compromised blood flow may be determined with any device in the art
useful for this purpose, such as a sphygmomanometer, blood pressure
meter, and the like. Compromised blood flow may also be determined using
techniques in the art useful for this purpose, such as the Color Doppler
technique, Pulsed Doppler, Power Doppler, Doppler ultrasound,
thermodilution, echo cardiography, plethysmography, and the like. Other
methods suitable for use in the present invention include cardiac
magnetic resonance imaging (MRI), computed tomography (CT) scan, cardiac
catheterization, chest CT, myocardial perfusion scan, radionucleotide
angiography, ultrafast CT scan, and the like.

[0157] Blood flow can also be monitored from an external position on the
vessel by using, for example, a flow probe.

[0159] All the materials and reagents required to determine the blood flow
in an artery or vein of an individual with suspected compromised blood
flow according to the present invention can be assembled together in a
kit, such kit includes at least UTP, a derivative thereof, or a salt
thereof as an active diagnostic ingredient, and instructions for the use
thereof in an individual with suspected compromised blood flow according
to any of the methods described herein.

[0160] In the above test kit, the reagents may be supplied from storage
bottles or one or more of the test tubes may be prefilled with the
reagents or controls.

[0161] The components of the kit, particularly UTP, a derivative thereof,
or a salt thereof, may be provided in dried or lyophilized forms. When
reagents or components are provided as a dried form, reconstitution
generally is by the addition of a suitable solvent. It is envisioned that
the solvent also may be provided in another container means.
Alternatively, UTP, a derivative thereof, or a salt thereof can be
provided in ready-to-use (pre-sterilized) form after reconstitution at
the use point. The UTP, a derivative thereof, or a salt thereof can also
be provided in suspended form, i.e., already suspended in the suitable
solvent.

[0162] The kits of the present invention also will typically include a
means for containing the reagents such as vials or tubes in close
confinement for commercial sale such as, e.g., injection or blow-molded
plastic containers into which the desired vials are retained.

[0163] The kits will also comprise a set of instructions on how to
determine suspected compromised blood flow according the methods of the
invention.

[0164] Different kits are provided with components, reagents and
instructions suitable for the preferred modes of delivery described
herein above, including delivery via continuous intravenous infusion, in
situ infusion, intracoronary infusion, drip infusion, intracoronary bolus
injection, a guiding catheter, or an IC micro catheter. Guiding
catheter(s) and/or microcatheter(s) may also be provided with the kits.

[0166] MPI is a form of functional cardiac imaging that may be used for
the diagnosis of ischemic heart disease. The underlying principle is that
under conditions of stress, diseased myocardium receives less blood flow
than normal myocardium. MPI is one of several types of cardiac stress
tests.

[0167] A cardiac specific radiopharmaceutical, such as
99mTc-tetrofosmin (Myoview, GE healthcare) or 99mTc-sestamibi
(Cardiolite, Bristol-Myers Squibb) is administered. Following this, the
heart rate and coronary blood flow is raised to induce myocardial stress
by systemic infusion of UTP, a derivative thereof, or a salt thereof.

[0168] SPECT imaging performed after stress reveals the distribution of
the radiopharmaceutical, and therefore the relative blood flow to the
different regions of the myocardium. Diagnosis is made by comparing
stress images to a further set of images obtained at rest (the reference
value). As the radionuclide redistributes slowly, it is not usually
possible to perform both sets of images on the same day, hence a second
attendance is required 1-7 days later.

[0169] However, if stress imaging is normal, it is unnecessary to perform
rest imaging, as it too will be normal--thus stress imaging only is
normally performed.

[0170] MPI has been demonstrated to have an overall accuracy of about 83%
(sensitivity: 85%; specificity: 72%) and is comparable with (or better
than) other non-invasive tests for ischemic heart disease.

Example 2

Local Infusion of UTP Via Guiding Catheter in the Coronary Arteries in
Humans with Coronary Artery Disease

[0171] The study was performed in 23 patients undergoing elective coronary
arteriography (CAG) due to repetitive episodes of typical ischemic chest
pain or in patients with recent Non-ST Elevated Myocardial Infarction
(>2 days from entry into study). In eligible patients with uni- or
multivessel coronary artery disease, lesions with stenosis of at least
50% of their diameter and that were thought to require PCI on the basis
of angiographic appearance and clinical data were identified.

Dose/Response Protocol

[0172] In the first part of the experiment, FFR and Coronary Flow
(velocity, CFR) were measured after the induction of coronary hyperemia
by a continuous intracoronary infusion of UTP (Jena Bioscience GmbH,
Germany) and adenosine in random order in a guide catheter (5F catheter,
Cordis Corp.). Two UTP doses of 240 and 360 μg/min were tested. UTP
was prepared by dissolving 100 mg of UTP trisalt (Sigma Aldrich) in 50 ml
isotonic NaCl. Of this 50 ml solution, 40 ml was filter sterilized and
added to sterile NaCl solution to a final volume of 500 ml, providing a
0.16 mg/ml concentration. 8-10 50 ml syringes of this solution can be
prepared (2 syringes per FFR study). 3 ml/min=480 μg/min=0.9
μmol/min. Subjects thus received either 1.5 ml/min of UTP infusion or
2.25 ml/min of UTP infusion for a total of 3 minutes each. Patients also
received adenosine (Sigma Aldrich) in the same eqipotent concentration.

[0173] All measurements were performed on at least 2 separate occasions to
achieve a reproducible result with a mean value calculated. After each
measurement, care was taken that APV returned to baseline before the
administration of the next dose. For all measurements using both drugs
and both routes of administration, changes in heart rate, blood pressure,
and ECG were recorded.

[0174] In the second part of the experiment, a full dose response curve
was generated for a gradual incremental increase in continuous UTP
infusion while both CFR and FFR were measured simultaneously prior to
percutaneous coronary intervention (PCI). Hyperemia was induced at 80
μg/min of continuous intracoronary UTP and adenosine, and the flow
reserve values were compared (Table 1).

[0175] In the third part of the experiment, both CFR and FFR were measured
simultaneously after percutaneous coronary intervention (PCI). Hyperemia
was induced by either 240 or 360 μg/min of continuous intracoronary
UTP and equipotent adenosine, and the flow reserve values were in some
patients compared with the hyperemic response of a complete, proximal
coronary occlusion for 30 s.

Calculations of Fractional and Coronary Flow Reserve

[0176] FFR is defined as the ratio of hyperemic flow in a stenotic artery
to the hyperemic flow in the same artery if there was no stenosis
present. FFR therefore expresses maximum hyperemic blood flow in a
stenotic vessel as a fraction of its normal value. FFR can be calculated
from intracoronary pressure measurements obtained during maximal
hyperemia by the following equation:
FFR=Pa-Pv/Pa-Pv-Pa/Pa, where Pa is the
mean proximal coronary pressure (mean aortic pressure), Pd is the
mean distal coronary pressure, and Pv is the mean central venous
pressure. The coronary flow (velocity) reserve is the ratio of maximum to
baseline hyperemic coronary flow velocity and is used as a surrogate for
CFR. Using the Volcano Combomap®, APV throughout the cardiac phase
was measured and CFR calculated from
APV.sub.(hyperernia)/APV.sub.(basal).

Results

[0177] The results of this experiment are shown in FIGS. 1-4 and 8.

[0178] Comparison of IC adenosine and IC UTP continuous adenosine infusion
in a dose response curve of six patients:

[0179] It is clear that at any given level of continuous UTP infusion, the
FFR value is lower in comparison to the same equipotent level of
adenosine. The lowest FFR level using adenosine is 0.81±0.11, however
the lowest level for UTP is 0.74±0.13; (P<0.05). As stated, the
doses of adenosine and UTP are different numerically because the
preparations have been made to be equipotent.

[0180] The times to induction of maximal hyperemia in all IC continuous
UTP infusion rates were shorter than that of IC continuous adenosine
infusion. As shown in all 23 patients, FFR was significantly decreased
with IC 240 μg/min infusion of UTP in comparison to adenosine infusion
(p=0.003) (FIG. 1). Consistent with this, Pd, or mean distal
coronary pressure, was also significantly decreased with UTP infusion
compared to adenosine infusion.

[0181] It has previously been shown that IC continuous adenosine infusion
is more effective in inducing maximal coronary hyperemia than IV
continuous adenosine infusion [8]. Particularly early induction of
optimal coronary hyperemia has a great advantage in repetitive
measurement of FFR. However, in comparison to UTP, adenosine is not as
vasodilatatorily potent presumably due to differences in activated
receptor types, affinity for the respective receptors, and amounts
infused. Thus, although the IC adenosine continuous infusion method for
FFR measurement is an accepted standard method for inducing maximal and
steady-state coronary hyperemia, it does not induce maximal hyperemia
when compared with UTP administration.

[0183] The optimal dose of UTP for achieving maximal coronary hyperemia by
our guide catheter method was UTP in concentrations of 80-400 μg/min.
Intracoronary UTP infusion at these doses can induce more rapid and more
potent coronary hyperemia than previous methods compared with equipotent
IC adenosine doses which are more potent than the standard IV adenosine
concentration of 140 μg/min. No further decrease in FFR was observed
after IC UTP infusion at >360 μg/min.

[0184] We conducted this study in most patients where FFRs were present in
the gray zone between 0.76 and 0.80 assessed by IC bolus adenosine
injection, but where IC infusion of UTP lowered the FFR even further due
to bigger post stenotic vasodilatation. The preferred infusion rate for
inducing maximal hyperemia by the IC continuous UTP infusion method is
thus ˜80 to 400 μg/min.

[0185] The results collectively demonstrate that IC continuous infusion of
UTP is safe and useful for inducing optimal coronary hyperemia without
any additional procedure; IC UTP infusion is a more potent vasodilatator
than IC adenosine infusion in equipotent concentrations; while previous
studies have shown that IC Ado ═IC ATP infusion>IV adenosine
infusion, the findings of the present study suggest that
UTP>adenosine=ATP; and IC UTP infusion is not associated with adverse
effects in contrast to IC or IV adenosine or ATP infusion.

Example 3

Local Infusion of UTP Via a Microcatheter in the Coronary Arteries in
Humans with Coronary Artery Disease

[0186] As described previously, inducing stable maximal coronary hyperemia
is essential for measurement of fractional flow reserve (FFR). This
second experiment in a similar patient group evaluated the differential
efficacy of intracoronary (IC) continuous adenosine infusion vs. IC
continuous uridine triphosphate (UTP) infusion via a microcatheter for
inducing steady state maximal coronary hyperemia. The present study was
designed to evaluate the safety and effectiveness of the equimolar
concentrations of UTP vs. adenosine infusion for use in the FFR method.
Time to achievement of steady-state, impact of maximal hyperemia (lowest
FFR) for the different compounds and side effects were recorded in 10
patients with intermediate coronary lesions. FFR was measured
consecutively by IC continuous adenosine or UTP infusion using a
microcatheter to bypass the proximal pressure transducer. The IC
microcatheter (Progreat Microcatheter System, Terumo, Japan) used was
positioned at the coronary ostium, and FFR was then measured by
increasing IC continuous adenosine or UTP infusion rates in equimolar
concentrations from 10 to 400 μg/min via the microcatheter.

[0187] After femoral catheterization, coronary angiography was performed
with the standard femoral approach. Heparin was administered according to
standard procedures. Intracoronary nitroglycerin (0.2-0.3 mg) was
administered before the control angiograms were made in the microcatheter
study. Heart rate and arterial pressure were continuously monitored
throughout the procedure. After a guiding catheter (5F catheter, Cordis
Corp.) without side holes was positioned at the coronary ostium, coronary
angiograms were obtained from multiple projections. Quantitative coronary
analysis was performed by using an independent analyzer blinded to the
results of FFR using a computer-assisted, automated computerized
edge-detection algorithm (Siemens Medical System). The external diameter
of the contrast-filled catheter was used as a calibration standard.
Minimal luminal diameter, vessel diameter of the reference segment, and
the percent diameter stenosis at end diastole were measured from the
worst-view trace. After coronary angiography, a 0.014-in. pressure wire
(Combowire, Volcano Corporation, US) was advanced distally to the
stenosis through a 6F guiding catheter. In the microcatheter study, an IC
bolus injection of nitroglycerin (0.2-0.3 mg) was given before advancing
the pressure wire through the stenosis in order to avoid any
mechanically-induced coronary vasoconstriction. The pressure wire was
externally calibrated and then advanced to the distal tip of the
catheter. It was verified whether equal pressure was recorded at both the
catheter and the pressure wire. The pressure wire was advanced through
the coronary catheter, introduced into the coronary artery, and
positioned distal to the stenosis.

[0188] Distal coronary pressure (Pd) and proximal coronary pressure
(Pa) were measured at baseline and at maximal hyperemia (adenosine
vs. UTP) simultaneously. Fractional flow reserve was calculated by
dividing mean Pd by mean Pa during maximal hyperemia. The time
to optimal vasodilatation (time needed to reach>90% of the minimal
value of Pd/Pa after administration of the adenosine or UTP)
was computed in order to assess whether procedure time was prolonged. The
hyperemic stimuli were given as follows: an IC continuous infusion of
adenosine or UTP in incremental doses of 10, 20, 40, 80, 160, 240, 360
and 400 μg/min, in both the left and right coronary artery depending
upon lesion anatomy. The dosages were increased stepwise by IC continuous
infusion with the microcatheter where the stepwise dose increase was
recorded continuously without breaks. After each stepwise increase, FFR
and CFR were recorded automatically. The next hyperemic stimulus UTP vs
adenosine was given when Pa, Pd, and heart rate returned to
their baseline values.

[0189] As described above, the results showed that the fractional flow
reserve measured by the IC continuous UTP infusion method was
significantly lower than that of the IC continuous adenosine infusion
method. Also, induction time to optimal coronary hyperemia by our method
was also shorter than that by the IC continuous adenosine infusion
method. As stated above, the induction of optimal coronary hyperemia has
a great advantage in repetitive measurement of FFR and our method makes
it possible to measure FFR repetitively and easily within a short period
of time when compared with previous methods. None of the patients had
chest pain in the UTP group but nearly all felt angina in the adenosine
group due to P1 pain receptor stimulation. Also, some patients had a
transient second degree A-V block during IC adenosine infusion. There was
no difference in systemic blood pressure and heart rate during IC
continuous UTP or adenosine infusion. The IC UTP infusion method is thus
safe for inducing coronary hyperemia without any systemic complications
compared to the IC adenosine infusion method.

[0190] The optimal dose of UTP for achieving maximal coronary hyperemia
and thereby the lowest FFR by the UTP method was approximately 80
μg/min for UTP and 240 μg/min for adenosine (FIG. 5). However, no
intracoronary adenosine infusion produced as low FFR levels at any given
concentration as that achieved during UTP infusion, meaning that
adenosine, even at its highest concentration, rendered a higher FFR than
UTP. UTP above 80 μg/min did not further lower FFR, and must thus be
assumed to be the correct amount to be infused. However, given that some
patients may be more or less responsive to this dose depending upon their
number of effective receptors and distribution, it is recommended that
the starting dose should be around 50 μg/min and then increased
incrementally until the lowest level of FFR is achieved. In this manner,
the dose range can be individualized according to any given patient's
need for an optimal diagnosis.

[0191] All patients tolerated UTP without side effects, but nearly all
patients experienced side effects during the adenosine procedures. FFRs
measured by UTP infusion were significantly lower than those by IC
adenosine infusion (P<0.05). Intracoronary UTP infusion was also able
to shorten the time to induction of optimal and steady-stable hyperemia
which also lasted slightly longer (˜20 seconds) with UTP compared
to adenosine infusion.

[0192] These results collectively suggest that IC continuous UTP infusion
using an IC microcatheter may be safe and useful for inducing optimal
coronary hyperemia for the individual patient without any additional
procedure. There are no obvious contraindications or cautions to consider
since UTP carries no side effects; UTP is more receptor selective and has
a faster and slightly longer steady state; UTP produces maximal
hyperemia, which is close to post occlusion hyperemia, thus allowing for
a more accurately estimate of maximal coronary blood flow and rendering a
more precise FFR calculation.

[0193] Not only can UTP be used in all patients following normal
guidelines for FFR use, it can be further extended to include those
patients who have contraindications to the use of adenosine and who for
this reason would not normally be FFR tested.

Example 4

Systemic Infusion of UTP in Pigs for Using UTP as a Coronary, Renal or
Peripheral Dilator in Diagnostic Hyperemic Methods

[0194] In the present study, the hemodynamic response in the pulmonary and
systemic circulation was tested, as well as the effect of central
intravenous infusions of ATP, ADP, ADO, and UTP on the heart.

[0195] Infusion rates were aimed at exerting a pronounced systemic
response in the lowering of arterial blood pressure, but avoiding a total
circulatory collapse.

Methods: 10 healthy female pigs (Department of Experimental Medicine and
Surgery, University of Copenhagen, Denmark) bred as a combination of the
Danish Landrace (1/3) and Yorkshire (2/3), with a medium weight of
41±2 kg were investigated.

[0196] Mean arterial blood pressure (MAP) was obtained from the catheter
in arcus aorta with the transducer (Pressure Monitoring Kit, Baxter,
Deerfield, Ill., USA) positioned at the level of the heart. The left
femoral artery was subsequently exposed and an ultrasound doppler probe
attached for peripheral femoral artery flow measurements (CM-4000,
Cardiomed, Norway). MAP, HR and peripheral flow were monitored and data
continuously collected using a PowerLab system (Adinstruments,
Australia). Cardiac Output (CO) was determined in triplicates by
thermodilution, following an injection of 10 ml of cooled saline
solution. Blood samples were withdrawn from catheters in aorta and the
right atrium at baseline and at steady state when MAP had decreased
maximally or by ˜50%. Calculated variables were: stroke volume of
the heart (SV=CO/HR), pulmonary vascular resistance (PVR=(MPAP-PCWP)/CO),
systemic vascular resistance (SVR=(MAP-MRAP)/CO), leg vascular
conductance (LVC=LBF/(MAP-MRAP)), and arterial (aorta) and mixed venous
(pulmonary artery) oxygen content (Hb*1.34*O2 saturation). In the
calculation of LVC, MRAP was used as an estimate of mean femoral venous
pressure (MFVP), as MRAP was found in a series of measurements to equal
MFVP, as estimated in 10 supine pigs. As a potential estimate for
myocardial oxygen demand the rate pressure product (RPP=MAP*HR) was also
calculated.

Drug infusions: Each animal was administered the nucleotides ATP, UTP,
ADP (Sigma, St. Louis, Mo., USA) and ADO (Item Development AB, Stocksund,
Sweden), through the internal jugular vein in the right atrium, in random
order following blinded allocation. The nucleotides and ADO were prepared
by dissolving in saline so as to achieve the target concentration of ATP
(40±0.5 μmol/min), ADP (43±8 μmol/min), UTP (up to 1,600
μmol/min), and ADO (73±7 μmol/min). The nucleotides were infused
in increasing dosages at 2 min intervals aiming at reducing MAP by
maximally ˜50%. Thereafter, that specific infusion rate was
maintained for ˜3-5 min. After each intervention the animal rested
for ˜30 min, allowing resting cardiovascular variables to be
reestablished. Thereafter, a new cycle of nucleotide infusion was
administered. In the present study we aimed to allow the drugs to reduce
MAP by ˜50%, but without inducing a hemodynamic and circulatory
collapse, to simulate a pronounced state of systemic vasodilation, as may
be observed for instance in a shock condition.

Results

Infusion Rates

[0197] There were no significant differences in any of the baseline
conditions before infusions of ATP, ADP, ADO or UTP (FIGS. 6 and 7). At
target MAP, corresponding to a ˜50% reduction in baseline MAP, ATP
and ADP were infused at similar rates (40.2±0.5 and 43.2±7.7
μmol/min, p=ns), whereas ADO was infused at a higher rate (72.7±6.6
μmol/min, p<0.05). A further increase in ATP, ADP and ADO infusion
rate, in contrary to UTP, could lower MAP below the target ˜50%
reduction in MAP, requiring careful monitoring during dosage titration.
In an attempt for UTP to reach ˜50% reduction in MAP, UTP infusion
rate was increased as high as 1.600 μmol/min in two animals. However,
UTP infusion did not decrease MAP more than ˜35%. This reduction
was also obtained at much lower UTP infusion rates (86.5±18.2
μmol/min). Since an increase in the dosage of UTP did not decrease MAP
further in those animals, the UTP infusion rate was not increased beyond
this dose in the remaining experiments, when this drop in MAP was reached
(FIG. 7a).

[0198] During infusion of ATP, ADP and ADO, MAP was lowered by
47.4±1.7, 48.4±1.2 and 47.2±1.5%, respectively, from stable
baseline values (p<0.05) (FIG. 7a). However, during UTP infusion, MAP
was only lowered by 35.0±3.2% (p<0.05) (FIG. 7a). Furthermore ATP,
ADO and UTP increased CO by 35.1±6.9, 31.4±9.9 and 72.5±15.2%,
respectively (p<0.05) (FIG. 7b). ADP infusion did, however, not alter
CO (p=ns). The CO increase during UTP infusion was furthermore greater
than the increase in CO during infusion of ATP and ADO (p<0.05). In
addition, ATP, ADP and UTP increased HR with 23.0±5.7, 26.6±4.6 and
51.1±9.0%, respectively, from stable baseline values (p<0.05) (FIG.
7c). ADO however, did not increase (p=ns) HR. The increase in HR was also
greater for UTP than for the other nucleotides (p<0.05). ATP, ADO and
UTP infusion did not significantly change SV (p=ns) (FIG. 7d). However,
ADP infusion decreased SV by 21.1±5.6% (p<0.05).

Nucleotides and their Effects on Vascular Resistance, Conductance and
Blood Flow.

[0199] ATP and ADO infusion decreased PVR by 37.8±4.9 and 34.3±2.6%,
respectively, from stable baseline values (p<0.05) (FIG. 7a). There
was no significant difference (p=ns) in the PVR change between the ATP
and ADO infusions. UTP did not significantly alter (p=ns) PVR. On the
contrary, ADP markedly increased PVR by 156.7±38.3% (p<0.05). PVR
furthermore rose early in the ADP titration procedure even during low
infusion rates and continued to increase dose dependently to 7.3±1.2
Wood Unit (p<0.05).

[0200] ATP, ADP, ADO and UTP infusion all decreased SVR by 61.6±2.1,
49.5±2.0, 59.0±3.0 and 62.9±2.6%, respectively, from stable
baseline values (p<0.05) (FIG. 3b). There was no significant
difference (p=ns) in the SVR change between trials. ATP, ADP and ADO
decreased LBF by 22.7±4.2, 34.9±10.2 and 19.4±10.7%,
respectively, from stable baseline values (p<0.05) (FIG. 6b). UTP
however increased LBF by 53.7±17.8% (p<0.05). There was no
difference (p=ns) in the change in LBF between these trials (FIG. 6c).
LVC showed a tendency to increase for all nucleotides, but this increase
was only significant for ATP, ADO and UTP (p<0.05); with an increase
by 44.7±8.7, 56.4±12.9 and 150.0±17.1%, respectively, from
stable baseline values (p<0.05). UTP increased LVC more than the other
nucleotides (p<0.05).

The Vasoactive Effect of ADP, ATP, UTP and Adenosine in the Leg (FIG. 6)

[0201] The vasodilator potency in the peripheral circulation revealed that
ADP>ATP=ADO. Thus ATP, ADP and ADO decreased LBF with 22.7±4.2,
34.9±10.2 and 19.4±10.7%, respectively, from stable baseline values
(p<0.05). UTP however increased LBF by 53.7±17.8% (p<0.05) (FIG.
6c), presumably because of a high increase in CO and a lesser degree of
systemic pressure reduction. LVC showed a tendency to increase for all
nucleotides, but this increase was only significant for ATP, ADO and UTP;
with an increase by 44.7±8.7, 56.4±12.9 and 150.0±17.1%,
respectively, from stable baseline values (p<0.05).

[0202] The study identified the unique differential properties of the
nucleotides ATP, ADP, ADO and UTP in the pulmonary, peripheral and
systemic circulation. This is the first study to simultaneous compare the
vasodilatory potency of nucleotides when infused in the right atrium.
Previous studies have shown that ATP, ADP, ADO and UTP all induce local
vasodilation when infused in the femoral artery and intravenous infusions
can mediate a decreases in MAP but none of these studies have compare the
relative potency of all of these nucleotides. With regards to the dose of
the nucleotides needed to reduce MAP by ˜50%, no difference in
potency was observed for ATP and ADP, despite different purinergic
receptor affiliation; where ADP predominantly stimulates P2Y1,
P2Y12 and P2Y13 receptors; and ATP predominantly stimulates
P2X, P2Y2, P2Y4 and P2Y11 receptors. ADO, predominantly
stimulating P1 receptors, was less potent than ADP and ATP, thus
requiring a higher infusion rate to produce the same decrease in MAP.
This makes it unlikely that the effect of ATP and ADP was due to the
dephosphorylated metabolites of these substances. UTP, stimulating
P2Y2 and P2Y4 receptors, was unable to produce the targeted
˜50% drop in MAP, due to a marked dose dependent rise in HR and CO.
These results differs from previous findings with intra-arterial
nucleotide infusions, where ATP and UTP were found to be equipotent, and
even more potent than ADO and ADP. This may be due to that the passage of
the nucleotides through the pulmonary and coronary circulation affects
the MAP response, differently from when infused intra-arterially.

[0203] The study also showed that UTP do not change PVR, despite an
increase in MPAP, as it was counterbalanced by an increase in CO, at an
unaltered PCWP. Previous studies have also suggested that ATP, but not
UTP, mediate vasodilation in the pulmonary artery, in the presence of a
functional endothelium. Although previous studies have shown that ATP and
UTP increase myocardial contractility, probably through P2X, P2Y2, P2Y6
and P2Y11-like receptors the present study only detected CO increases
during infusions of ATP, ADO and UTP; and to the greatest extent for UTP.
ATP, ADP and UTP all increase HR, whereas ADO do not change HR
significantly. UTP furthermore increases HR significantly more than ATP
and ADP.

[0204] Relevance for diagnostic use: These results collectively suggest
that when UTP is infused intravenously in the systemic circulation it:
increases cardiac output (CO) by ˜70% due to increases in HR, thus
resembling an exercise condition; it has a tendency to decrease rate
pressure product by ˜10%, thereby being safe for patients with
ischemia; and importantly does not produce arrhythmias (missed beats, VT,
SVT or AV nodal block). Furthermore, UTP increases leg blood flow by
˜50% presumably because of a higher increase in CO (such as during
exercise) and has a lesser degree of systemic pressure reduction compared
to other adenine compounds, making it suitable for use with indications
such as aorta stenosis, peripheral arterial disease (PAD), or kidney
stenosis with methods such as myocardial perfusion imaging, echo or MRI).
Furthermore, because it increases leg vascular conductance by ˜150%
(only ˜50% with adenosine), UTP is ideal for PAD diagnosis because
it mimics exercise-induced vasodilatation.

Example 5

[0205] The following data demonstrate that a 0.05 difference in FFR
between adenosine and UTP with a standard deviation of 0.15-0.17
represents a statistically significant difference (p=0.003).

[0206] If we were planning a study of a continuous response variable from
matched pairs of study subjects, the prior data indicate that the
difference in the response of matched pairs is normally distributed with
a standard deviation of 0.16. If the true difference in the mean response
of matched pairs is 0.05, we will need to study only 135 pairs of
subjects to be able to reject the null hypothesis that this response
difference is zero with probability (power) 0.95. The Type I error
probability associated with this test of this null hypothesis is 0.05.

[0207] The cut-off value for FFR is usually 0.8 for being indicative of a
treatment intervention being required (according to the set FFR value in
the FAME study). This means that if patients have a FFR>0.8, they can
be left untreated, however if the value is <0.8, they should be
subjected to a PCI with insertion of a stent or bypass surgery according
to the lesion anatomy For the guiding catheter study, a FFR set at
≦0.75 or ≦0.8 would require altered treatment regiments in
the below percentage of patients with:

[0208] Consequently, by using UTP in accordance with the present
invention, it would be possible to diagnose more people than by the known
use of adenosine regardless of the set cut-off value for the same
concentration.

[0209] As seen from the microcatheter study, a FFR set at ≦0.8
would require altered treatment regiments in the below percentage of
patients in the different concentration:

[0210] Although it may seem as if ado=UTP for the higher concentrations,
the estimate of FFR is always based on the lowest possible FFR, because
only at this point is there maximal hypermia which corresponds to the
correct perfusion pressure.

Example 6 (Prophetic)

Use of UTP, a Derivative Thereof, or a Salt Thereof to Diagnose Renal
Artery Stenosis

[0211] When a renal arterial stenosis is identified on an arteriogram,
intra-arterial systemic pressure can be measured continuously with a
transducer and a miniaturized pressure-gradient wire system
(PressureWire; St. Jude Medical or Volcano combo wire). Pressures can be
recorded using a fiber-optic pressure sensor located laterally and 3 cm
from the distal end. The basic principle is that the element modulates an
optical reflection with pressure-induced elastic movements. This pressure
wire thus replaces a standard 0.018-inch guide wire. After advancing a 4
to 7-F guiding catheter from the femoral artery to the ostium of the
renal artery, a "coronary" 0.014-inch wire is introduced into the guiding
catheter and moved to the ostium of the stenosis. A pressure gradient
across renal arteries can be assessed when combined with an infusion of
UTP, a derivative thereof, or a salt thereof to induce renal hyperemia.
Infusion of adenosine will, under these circumstances, constrict the
afferent arterioles, causing dose-dependent renal vasoconstriction,
whereas UTP produces the desired renal vasodilatation. After identical
pressure of the guiding catheter and the wire is confirmed at this
position, the stenosis can be traversed by means of the floppy-ended
wire, followed by the transducer. Dilation equipment can then be inserted
through the guiding catheter and across the stenosis, leaving the wire in
place. Immediately after the intervention, results can be tested using
another infusion of UTP, a derivative thereof, or a salt thereof. The
pressure gradient is thus measured with the wire before and during the
infusion.

[0212] Patients with a renal artery stenosis with a Pd/Pa ratio larger
than 0.90 can be considered hemodynamically insignificant, and it is
unlikely that renal angioplasty would be useful in such patients even
though percent diameter stenosis is larger than 50%. Conversely, renal
artery stenoses with a Pd/Pa ratio<0.90 should be considered
hemodynamically significant regardless of angiographic severity.
Furthermore, a combined catheter with pressure and UTP infusion could
ensure a local infusion of the compound to prevent systemic spill over in
patients.

[0213] All patent and non-patent references cited in the application are
hereby incorporated by reference in their entirety.